Profiling using protein x basophil array: current progress

Abstract

Background: Correlation between observed allergic clinical symptoms and specific IgE levels is poor in some cases. Current developments on basophil activation tests (BAT) are showing improved clinic relevance (compared to DBPCFC) than specific IgE alone. We are developing a two stages diagnostic device which combines the advantages of BAT with the numerical power of protein microarrays. Previously we have shown that a four immunoglobulin platform for simultaneous detection of IgM, A, G and E was feasible. We then demonstrated as proof-of-principle that human peripheral blood basophils and humanised basophil cell lines can bind to a protein array and that activation can be detected. Here we show an application of the basic platform (protein array) and discuss current stages of development of the basophil array system. Method: Microarrays containing proteins and extracts of food products, enterobacteria and inhaled allergens have been hybridised with sera from a retrospective longitudinal trial of children with clinically well-characterized cow’s milk allergy (n= 41) and control sera (n= 20) as previously described. The IgM, A, G and E levels have been determined and were analysed using chemometric multivariate routines. For the second stage (basophil cells), binding and incubation parameters to the array were experimentally optimised for three independent cell lines. Basophil cell activation markers involving Annexin V binding, Calcium influx and fluorescent inducible reporter systems have been compared to enzymatic degranulation tests. Result: The basic platform (protein array only) has shown that total IgG and IgA share similar specificity whilst IgM and IgE in particular are distantly related. The array has shown that four out of the 41 patients might have allergies other than milk origin. A good correlation (r2> 0.7) between dairy IgE and ImunoCAP, casein in particular, was observed. In the basophil system (2nd stage platform), measurement of Annexin V and calcium influx produced expected degranulation endpoints however, fluorescent reporter genes have shown higher sensitivity. The lack of stability of cells after successive passages is still a major issue. Conclusion: The basic platform (protein array) for the comprehensive profiling tools has produced qualitatively and quantitative reproducible results. The optimisation of the second component (basophil cells) of the profiling platform that is, reporter genes for key steps.